Acteoside ameliorates hepatic ischemia-reperfusion injury via reversing the senescent fate of liver sinusoidal endothelial cells and restoring compromised sinusoidal networks

Hepatic ischemia-reperfusion injury (HIRI), a common two-phase intersocietal reaction in liver surgery, typically leading to sustained liver dysfunction. During this process, liver sinusoidal endothelial cells (LSECs) are vulnerable to damage and exert senescence-associated secretory phenotype (SASP). However, how these SASP-LSECs secreted damage-associated molecular patterns (DAMPs) to impact the whole HIRI microenvironment and whether it can be reversed by therapeutics remains unknown. Here, we found that either HIRI surgery or hypoxia and reoxygenation (HR) stimulation forced LSECs into SASP and expressed HMGB1-dominated DAMPs, which were dramatically improved by acteoside (ACT). Additionally, hypoxic hepatocytes released excessive HMGB1 to LSECs and synergistically aggravated their SASP state. Mechanistically, HMGB1 bound with TLR3/TLR4 on LSECs, promoted the nuclear translocation of IRF1 and subsequent transcription of cxcl1 and Hmgb1, leading to the chemotaxis of neutrophils and accelerating immune damage in a vicious circle. Notably, ACT or HMGB1 siRNA effectively disrupted HMGB1-TLR3/4 interaction, leading to IRF1 inhibition and repairing LSEC functions, which was largely reversed by HMGB1 stimulation and IRF1-overexpressed liposomes with LSECs-targeted hyaluronic acid-derivative conjugated in mice. Collectively, ACT reversed the senescent fate of LSECs and restored sinusoidal networks by targeting HMGB1-TLR3/4-IRF1 signaling, thus providing protection against HIRI and offering the potential for new therapeutics development.


Serum biochemical measurements
The blood of mice was collected from the inferior vena cava and was centrifuged for 10 min at 1200g to obtain serum.Alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactic dehydrogenase (LDH), NO, and hyaluronic acid (HA) in the serum were measured by relative detection kits to evaluate the degree of HIRI in mice, according to the detailed manufacturer's specifications.

Histopathological examination
After being fixed in 4% paraformaldehyde and embedded in paraffin, liver specimens stained with hematoxylin and eosin (H & E) reagents were scanned and analyzed by super-resolution microscopy (Leica, Wetzlar, Germany).For transmission electron microscope (TEM) examination, liver tissues were immersed in specialized TEM formalin-free fixative solution, which was rapidly sliced and prepared for scanning to examine the hepatic sinusoid architectures.All images were recorded using Zeiss Libra 120 transmission electron microscopy (Carl Zeiss, Germany).

RNA isolation and quantitative RT-PCR.
The total RNA from different cells or HIRI mouse livers were isolated by TRIzol reagent and were reverse transcribed to cDNA.The expressions of the target genes were detected by SYBR Green quantitative real-time polymerase chain reaction.
Target gene expressions were calculated by their ratios to the housekeeping gene Hprt1.Further inquiries of primers for qPCR can be directed to the corresponding author.

Enzyme-linked immunosorbent assays (ELISA)
After setting up the standard curve, HMGB1 and C-X-C motif chemokine ligand 1 (CXCL1) from the cell culture medium and mouse serum was measured by different HMGB1 and CXCL1 ELISA kits, according to the described manufacturer's protocols.
The optical density of each well was determined using a microplate reader.

Flow cytometry analysis
Cell proliferation and apoptosis of LSECs were measured using AV/PI kit (Beyotime, Shanghai, China) following the manufacturer's instruction.Briefly, the primary LSECs were harvested by trypsin for AV/PI staining, then washed with PBS

Senescence associated β-galactosidase staining (SA-β-Gal)
SA-β-Gal staining kit (G1580) was obtained from Solarbio (Beijing, China) and the pre-heated SA-β-Gal solution was used for staining of aging hepatocytes and LSECs.Briefly, different hepatic cells in a 6-well plate were fixed with β-Gal formaldehyde solution for 15 min, washed with 1× PBS 3 times, and stained for 12h with X-Gal solution 1 ml.The slides were subsequently washed and mounted for microscope scanning and Image J was used for staining quantification.

5-Ethynyl-2'-deoxyuridine staining (EdU) incorporation and staining
The EdU staining kit (APEx BIO, USA) was used for evaluating the proliferation of hepatocytes and LSECs.Firstly, the 10× EdU buffer dissolving in dimethyl sulfoxide diluted with culture medium at 4 mM was prepared.Then, the cells in different groups were cultured in a 6-well plate and incubated with EdU buffer at a concentration of 2 mM for 2 h.After being washed twice and fixed in 4% formaldehyde for 15 min at room temperature, cells were incubated with 0.3% Triton X-100 diluting in 3% bovine albumin-PBS for 15 min and washed twice again.For the EdU click reaction, cells were incubated with the working buffer (including the 1× EdU reaction buffer, CuSO4, Cy3 azide, and 1× EdU buffer addictive) for 30 min in the darkness.After being washed for 2 times, cells were embedded with DAPI and observed under the confocal laser scanning microscopy (Olympus FV3000, Tokyo, Japan).

Transwell assay
For the transwell experiment of neutrophils recruited by LSECs, a total of 1 × 10 6 primary isolated neutrophils were seeded in the upper chamber of the transwell systems (0.4 μm) with 250μl 1% FBS DMEM medium, and 1 × 10 6 attached LSECs were added in the bottom chamber under HR process and treated with ACT (25, 50 and 100 μM) with 500ul 1% FBS DMEM medium for 6 h.After incubating for 12 h, the cells in the bottom were removed slightly with a cotton swab, while the cells on lower filter surfaces were fixed by the 4% formaldehyde and then stained with the 0.1% crystal violet solution for 30 min.The number of migrated cells was counted by an optical microscope (Carl Zeiss AG, Oberkochen, Germany).
and re-suspended by 200 μl 1× Binding Buffer with 5 μl FITC Annexin V and PI for 20 min in the dark at room temperature.Finally, AV/PI signals were visualized by a CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA).

Fig. S8 .
Fig. S8.The isolation and identification of primary neutrophils.(A) The flow chart